Effect of temperature and growth media on the attachment of Listeria monocytogenes to stainless steel

Listeria monocytogenes ATCC 19111 cultivated in nutrient-rich medium (brain heart infusion, BHI) or starved in minimal medium (10% filter sterilized pond water and 90% sterilized distilled water) were investigated for their initial attachment to austenitic stainless steel No. 4 with satin finish at 4 °C, 20 °C, 30 °C, 37 °C, or 42 °C. A droplet (10 μl) containing  10⁷ CFU/ml of L. monocytogenes suspended in BHI or minimal medium was placed on the stainless steel surface. After holding in saturated humidity for 3 h at the desired temperature the surface was washed and prepared for scanning electron microscopy (SEM). Using SEM, attachment of L. monocytogenes was determined by counting cells remaining on the surface. When L. monocytogenes cultivated in BHI were used, with the exception of the number of attached cells being lower at 42 °C than at 37 °C and 30 °C, the number of attached cells increased with increasing temperature (P < 0.05). When L. monocytogenes starved in minimal medium were used, the number of attached cells also increased with increasing attachment temperature (P < 0.05), but the number of attached cells at 42 °C was lower than that at the other temperatures. The attachment of L. monocytogenes to stainless steel surface was greater when cultivated in rich medium of BHI vs starved in the minimal medium.     

Effect of surface roughness and stainless steel finish on Listeria monocytogenes attachment and biofilm formation

The purpose of this study was to evaluate the effect of surface roughness (Ra) and finish of mechanically polished stainless steel (Ra = 0.26 +/- 0.05, 0.49 +/- 0.10, and 0.69 +/- 0.05 microm) and electropolished stainless steel (Ra = 0.16 +/- 0.06, 0.40 +/- 0.003, and 0.67 +/- 0.02 microm) on Listeria adhesion and biofilm formation. A four-strain cocktail of Listeria monocytogenes was used. Each strain (0.1%) was added to 200 ml of tryptic soy broth (TSB), and coupons were inserted to the mixture for 5 min. For biofilm formation, coupons with adhesive cells were incubated in 1:20 diluted TSB at 32 degrees C for 48 h. The experiment was performed by a randomized block design. Our results show that the level of Listeria present after 48 h of incubation (mean = 7 log CFU/cm2) was significantly higher than after 5 min (mean = 6.0 log CFU/cm2) (P < 0.01). No differences in initial adhesion were seen in mechanically finished (mean = 6.7 log CFU/cm2) when compared with electropolished stainless steel (mean = 6.7 log CFU/cm2) (P > 0.05). Listeria initial adhesion (values ranged from 5.9 to 6.1 log CFU/cm2) or biofilm formation (values ranged from 6.9 to 7.2 log CFU/cm2) was not significantly correlated with Ra values (P > 0.05). Image analysis with an atomic force microscope showed that bacteria did not colonize the complete surface after 48 h but were individual cells or grouped in microcolonies that ranged from 5 to 10 microm in diameter and one to three cell layers in thickness. Exopolymeric substances were observed to be associated with the colonies. According to our results, electropolishing stainless steel does not pose a significant advantage for food sanitation over mechanically finished stainless steel.     

Marked intra-strain variation in response of Listeria monocytogenes dairy isolates to acid or salt stress and the effect of acid or salt adaptation on adherence to abiotic surfaces

During food processing, and particularly in cheese manufacturing processes, Listeria monocytogenes may be exposed routinely to environments of low pH or high salt concentration. It has been suggested that these environmental conditions may contribute to bacterial adherence to abiotic surfaces and increased resistance to disinfection. In this study strains isolated from the environment of artisanal cheese-making dairies were used to investigate the behaviour of L. monocytogenes in response to acid and salt stress and clear differences between strains was observed. In planktonic culture, strains varied in resistance to low pH or high NaCl concentration and in the occurrence of an adaptive response to moderate acid or NaCl. There was dislocation in responses to salt and acid. Strains resistant, or adaptive, to acid were not resistant or adaptive to NaCl. The reverse also was observed. Exposure to moderate acid did not promote adherence to polystyrene but survival, at low pH or high NaCl concentration, of cells adherent to stainless steel was increased, even for strains that had no adaptive response planktonically, but the detail of these observations varied between strains. In contrast to acid adaptation, with some strains salt adaptation enhanced adherence of L. monocytogenes to polystyrene but this was not true for all strains. For some strains salt- or acid adaptation may enhance the survival of sessile cells exposed to hypochlorite disinfection.     

Monolaurin and acetic acid inactivation of Listeria monocytogenes attached to stainless steel.

Individual and combined antimicrobial effects of monolaurin and acetic acid on Listeria monocytogenes planktonic cells or stainless-steel-adherent cells were determined in order to evaluate cell viability during a 25-min exposure period at 25 degrees C. A 10(7)-colony-forming units (CFU)/ml population of planktonic cells was completely inactivated by the synergistic combination of 1% acetic acid with 50 or 100 microg/ml of monolaurin within 25 or 20 min, respectively. Either compound alone caused partial but incomplete inactivation within the same time periods. A population of 10(5) CFU/cm2 of 1-day adherent cells on stainless steel was completely inactivated within 25 min, but with the highest concentrations of the combined chemicals, i.e., 1% acetic acid and 100 microg/ml of monolaurin. The combined chemical treatment again synergistically produced greater inhibition. A 10(6)-CFU/cm2 population of 7-day adherent cells was not completely inactivated within 25 min of exposure, although counts did decline. The results demonstrate increased resistance of attached L. monocytogenes to acetic acid and monolaurin and show that resistance increased with culture age. Combinations of organic acids and monolaurin might be considered as sanitizers of food contact surfaces, but activities of such combinations are likely to be less than other commonly used sanitizers.     

Heat and acid tolerance of Listeria monocytogenes after exposure to single and multiple sublethal stresses

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.     

Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp

Contaminated surfaces of food processing equipment are believed to be a significant source of Listeria monocytogenes to foods. However, very little is known about the survival of Listeria in processing environments. In a mixed bacterial biofilm of L. monocytogenes and Flavobacterium spp., the number of L. monocytogenes cells attaching to stainless steel increased significantly compared to when L. monocytogenes was in a pure culture. The L. monocytogenes cells in the mixed biofilms were also recoverable for significantly longer exposure periods. On colonized coupons held at 15 degrees C and 75% humidity, decimal reduction times were 1.2 and 18.7 days for L. monocytogenes in pure and mixed biofilms, respectively. With increasing exposure time, the proportion of cells that were sublethally injured (defined as an inability to grow on selective agar) increased from 8.1% of the recoverable cell population at day 0 to 91.4% after 40 days' exposure. At 4 and -20 degrees C, decimal reduction times for L. monocytogenes in pure culture were 2.8 and 1.4 days, respectively, and in mixed culture, 10.5 and 14.4 days, respectively. The enhanced colonization and survival of L. monocytogenes on "unclean" surfaces increase the persistence of this pathogen in food processing environments, while the increase in the percentage of sublethally injured cells in the population with time may decrease the ability of enrichment regimes to detect it.     

Role of sigma B factor in the alkaline tolerance response of Listeria monocytogenes 10403S and cross-protection against subsequent ethanol and osmotic stress

Many of the considerable abilities of Listeria monocytogenes to persist and grow in a wide range of adverse environmental conditions are thought to be at least partly under the control of the alternative sigma factor (sigmaB), encoded by the sigB gene. However, little is known about the role of this master regulon in the impressive ability of Listeria to persist and grow under conditions of alkaline pH. In this study, Northern blot analysis of parent Listeria mRNA revealed that alkali adaptation (pH 9.5 for 1 h) significantly increased the expression of sigB-derived mRNA. The study included a comparison of the relative survival of mid-exponential populations of adapted and nonadapted parent type (sigmaB expressing) and mutant (not sigmaB expressing, deltasigB) Listeria strains during subsequent alkaline (pH 12.0), osmotic (25% NaCl, wt/vol), or ethanol (16.5%) stress. Alkali-adapted parent strains were more resistant to pH 12.0 than were adapted deltasigB type strains, but both alkali-adapted parent and deltasigB strains were more resistant to pH 12.0 than were nonadapted strains. Alkali-adapted parent strains were more resistant to osmotic stress than were adapted deltasigB type strains. No significant differences in viability were observed between alkali-adapted parent and deltasigB strains after ethanol stress, suggesting that cross-protection against osmotic stress is mediated by sigmaB whereas cross-protection against ethanol is sigmaB independent. Overall, alkali-induced cross-protection against osmotic and ethanol challenges may have serious implications for food safety and human health because such stress conditions are routinely used as part of food preservation and surface cleaning processes.     

Variation of antibiotic resistance in Salmonella Enteritidis,Escherichia coli O157:H7, and Listeria monocytogenes after exposure to acid,salt, and cold stress

Bacteria with antibiotic-resistant could seriously threaten to human health, increasing the treatment cost for infections and negatively affecting treatment outcomes. Stress adaptation is one possible mechanism for the acquisition or enhancement of antibiotic resistance in bacteria as a result of cross-protection. In this study, the effects of acid, salt, and cold stress on the antibiotic resistance of Salmonella Enteritidis, Listeria monocytogenes, and Escherichia coli O157:H7 were investigated using the disc diffusion method. For S. Enteritidis, acidic growth conditions increased resistance to ciprofloxacin and erythromycin (p < .05), and addition of 4% NaCl to growth media decreased resistance to chloramphenicol (p < .05). Irrespective of pH and the NaCl concentration of the growth medium, refrigerated E. coli O157:H7 showed increased resistance to amoxycillin, ciprofloxacin, gentamicin, streptomycin, and erythromycin (p < .05). Acid-adapted L. monocytogenes showed decreased the resistance to amoxycillin, ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, streptomycin, and tetracycline (p < .05). In conclusion, prolonged exposure of foodborne pathogens to acid, salt, and cold stress alters their antibiotic resistance. However, the effect of acid, salt, and cold stress on bacterial antibiotic resistance depend on both the bacterial species and the specific antibiotic. Therefore, multiple factors need to be considered for a foodborne antimicrobial resistant risk assessment.     

The resistance to detachment of dairy strains of Listeria monocytogenes from stainless steel by shear stress is related to the fluid dynamic characteristics of the location of isolation

Strains of Listeria monocytogenes isolated from artisanal Portuguese cheese-making dairies were divided into two categories on the basis of the locations from which they were isolated: strains from dynamic locations were those that were habitually exposed to flowing liquids during the process of cheese-making, whereas those from static locations were rarely, if ever, exposed to the shear stresses generated by liquid flows. The strength of attachment to stainless steel discs of all of these strains was obtained using a radial flow chamber. Initial attachment strengths to stainless steel (after a 0.5 h contact time) of L. monocytogenes strains were greater for the 5 isolates from surfaces exposed to flow (dynamic isolates) than for most (3 out of 4) of those that were not (static isolates). After a 24 h contact time, attachment strength of all isolates reached similar levels. These results suggest that strains having high initial attachment strength are more likely to persist on surfaces exposed to flow than strains having low initial attachment strength. The numerical values of shear forces obtained could prove useful in the rational design of cleaning and decontamination procedures in food processing facilities.     

Effects of the growth procedure on the surface hydrophobicity of Listeria monocytogenes cells and their adhesion to stainless steel.

The aim of this study was to examine the physicochemical surface properties and the ability to adhere to stainless steel of three strains of Listeria monocytogenes after different cultivation procedures. To this end, bacteria were cultivated at 37 degrees C after storage at two frequently used temperatures (4 degrees C or -80 degrees C) and were then transferred into the liquid medium (trypticase soy broth supplemented with 6 g liter(-1) of yeast extract, pH 7.3) between one and four times. In addition, the influence of supplementing the growth medium with lactic acid was explored, this organic acid being representative of both the dairy and cured meat industries. The hydrophobic/hydrophilic and electron-acceptor/electron-donor characteristics of the strains were evaluated by the microbial adhesion to solvents method. Using this technique, we recorded an increase in the hydrophobic properties of one strain stored at 4 degrees C, with an increasing number of transfers in the media (P < 0.05). Another plant-isolated strain appeared more hydrophobic and stuck better to stainless steel when cells were stored at 4 degrees C rather than at -80 degrees C. Preculturing L. monocytogenes in a lactic acid-supplemented medium increased the affinity of microbial cells to solvents and the bacterial attachment to stainless steel (P < 0.05).     

Effect of biofilm dryness on the transfer of Listeria monocytogenes biofilms grown on stainless steel to bologna and hard salami

Listeria monocytogenes continues to be a major cause of class I food recalls in the United States. Very little is known about its transfer and cross-contamination in processing scenarios. The objective of this study was to evaluate the effect of hydration level on L. monocytogenes biofilms grown on stainless steel and its effect on the biofilm transfer to foods. Biofilms were grown on stainless steel in diluted tryptic soy broth 1:20 for 48 h at 32 degrees C. After this, biofilms were equilibrated over saturated salt solutions at 20 degrees C for 24 h (94, 75, 58, and 33% relative humidity; % RH) prior to transferring. Transfer experiments were conducted from inoculated stainless steel to bologna and hard salami at a constant pressure (45 kPa) and time (30 s) with a universal testing machine. The experiment was designed with a factorial design 4 x 2 (biofilms equilibrated at 4% RH and two foods) and duplicated every day, and the whole experiment was repeated nine times. The results were analyzed with an analysis of variance by SAS Statistical Analysis Software. Our results showed that more bacteria were transferred to bologna (mean efficiency of transfer [EOT] = 3.0) than to hard salami (mean EOT = 0.35, P < 0.01). As biofilms became drier, the transfer of Listeria from stainless steel to both foods increased (P < 0.05). The EOT increased from 2 to 3.8 and from 0.2 to 0.51 upon transfer when drying the biofilm for bologna and hard salami, respectively. This study may be an indication that as biofilms were dried, the cell-cell and cell-surface interactions became weaker, and bacterial transfer increased. This phenomenon was enhanced in foods containing higher water activity levels. We hypothesize that this increased in transfer was due to the presence of capillary forces in the food.     

Bactericidal activity of strong acidic hypochlorous water against Escherichia coli O157:H7 and Listeria monocytogenes in biofilms attached to stainless steel

Food Science and Biotechnology - This study aims to investigate the bactericidal activity of strong acidic hypochlorous water (SAHW) against Escherichia coli O157:H7 and L. monocytogenes in...     

Evaluation of growth/no growth interface of Listeria monocytogenes growing on stainless steel surfaces, detached from biofilms or in suspension, in response to pH and NaCl

The present study aimed to describe the growth/no growth interface of Listeria monocytogenes at three potential states of growth in industrial environments, namely attached, (Att), detached (Det) from a biofilm, or in a planktonic state (suspended; Plan). A 3-strain composite of L. monocytogenes cells was left to colonize stainless steel (SS) surfaces in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 20 °C for 72 h and then transferred to TSBYE at 30 different pH and NaCl concentrations, which were renewed every two days during incubation at 10 °C. Survival of attached population was observed at optimal conditions (pH 7.2, a(w) 0.996), whereas at 4.5-8.0% salt and/or pH<6.0, reduction of attached population on SS surfaces was observed. PFGE patterns showed that 91% of the cells colonizing the SS coupons after 30 days, at any pH and a(w) conditions, belonged to a single strain. Furthermore, the change in the probability of a single cell to initiate growth (P(in)) over time, as well as the number of cells needed (CN) for growth initiation of planktonically growing Plan and Det L. monocytogenes cells were evaluated based on MPN Tables. An ordinary logistic regression model was also used to describe the growth/no growth interface of varying inoculation levels (from <10 to 10(4)CFU/ml) of Plan and Det cells in response to pH and a(w). Although both cell types demonstrated similar growth limits at populations of 10(2)-10(4)CFU/ml, higher numbers of Det than Plan cells were needed (CN) in order to initiate growth at low a(w) and pH. Individual Plan cells reached higher maximum levels of probability of growth initiation (P(max)) and had shorter times to reach P(max)/2 (t(au)), compared to their Det counterparts. Data on growth potential of cells in suspension, attached or detached status, may assist in ranking the risk from different sources of contamination. In addition, they may establish the link between the behavior of L. monocytogenes in foods and its origin from the processing plant. The latter link is important component of biotraceability.     

Effect of acid adaptation on growth during storage at 10 degrees C and resistance to simulated gastric fluid of Listeria monocytogenes inoculated onto bologna formulated with or without antimicrobials

The fate of acid-adapted and nonadapted Listeria monocytogenes inoculated onto bologna slices (formulated with or without antimicrobials) was examined during storage and after exposure to in vitro gastric challenge. Bologna slices formulated with no antimicrobials (control), 3% sodium lactate (SL), or 1.8% SL plus 0.25% sodium diacetate (SD) were inoculated (2 log CFU/cm2) with a 10-strain composite of acid-adapted or nonadapted L. monocytogenes strains. Growth or survival of the two inocula on bologna was evaluated during vacuum-packaged storage (10 degrees C) for up to 36 days. Survival of previously acid-adapted or nonadapted L. monocytogenes on stored bologna exposed to simulated gastric fluid (adjusted to pH 1.0 with HCl) for 20, 40, and 60 min also was determined. As expected, inclusion of antimicrobials in the product formulation inhibited growth of L. monocytogenes during storage of vacuum-packaged bologna compared with growth on control samples. Acid adaptation of L. monocytogenes prior to product inoculation did not affect subsequent survival or growth on bologna or resistance to simulated gastric fluid (P > 0.05). Survival of L. monocytogenes exposed to simulated gastric fluid during storage increased with product age, growth phase of the cells, and possibly age of the cells, particularly for control samples (no antimicrobials), in which the pathogen grew uninhibited to approximately 6 log CFU/cm2 by day 8 of storage. Inhibition of L. monocytogenes growth on product formulated with antimicrobials was associated with only sporadic and small numbers of survivors following exposure of these samples to simulated gastric fluid, especially in samples stored longer. However, cell numbers in these treatment groups before the gastric challenge did not exceed 3.8 log CFU/cm2. Inhibition of growth on product with antimicrobials precluded detection of survivors resistant to the effects of simulated gastric fluid.     

Biofilm formation and sporulation by Bacillus cereus on a stainless steel surface and subsequent resistance of vegetative cells and spores to chlorine, chlorine dioxide, and a peroxyacetic acid-based sanitizer

Biofilm formation by Bacillus cereus 038-2 on stainless steel coupons, sporulation in the biofilm as affected by nutrient availability, temperature, and relative humidity, and the resistance of vegetative cells and spores in biofilm to sanitizers were investigated. Total counts in biofilm formed on coupons immersed in tryptic soy broth (TSB) at 12 and 22 degrees C consisted of 99.94% of vegetative cells and 0.06% of spores. Coupons on which biofilm had formed were immersed in TSB or exposed to air with 100, 97, 93, or 85% relative humidity. Biofilm on coupons immersed in TSB at 12 degrees C for an additional 6 days or 22 degrees C for an additional 4 days contained 0.30 and 0.02% of spores, respectively, whereas biofilm exposed to air with 100 or 97% relative humidity at 22 degrees C for 4 days contained 10 and 2.5% of spores, respectively. Sporulation did not occur in biofilm exposed to 93 or 85% relative humidity at 22 degrees C. Treatment of biofilm on coupons that had been immersed in TSB at 22 degrees C with chlorine (50 microg/ml), chlorine dioxide (50 microg/ml), and a peroxyacetic acid-based sanitizer (Tsunami 200, 40 microg/ml) for 5 min reduced total cell counts (vegetative cells plus spores) by 4.7, 3.0, and 3.8 log CFU per coupon, respectively; total cell counts in biofilm exposed to air with 100% relative humidity were reduced by 1.5, 2.4, and 1.1 log CFU per coupon, respectively, reflecting the presence of lower numbers of vegetative cells. Spores that survived treatment with chlorine dioxide had reduced resistance to heat. It is concluded that exposure of biofilm formed by B. cereus exposed to air at high relative humidity (> or =97%) promotes the production of spores. Spores and, to a lesser extent, vegetative cells embedded in biofilm are protected against inactivation by sanitizers. Results provide new insights to developing strategies to achieve more effective sanitation programs to minimize risks associated with B. cereus in biofilm formed on food contact surfaces and on foods.